Cyanide (CN = HCN + CN-) is a ubiquitous environmental toxicant. Despite a plethora of studies, little is known of its potential to cause adverse health effects. CN exposure is normally determined from blood by a GC-MS headspace assay. However, CN is rapidly cleared from this compartment (t1/2 in blood is <1 hr), and it is common for several half-lives to have passed before blood samples are drawn for analysis. Consequently, assessment of acute human CN exposure in high-risk populations is highly problematic. Prior studies by us showed that CN reacts with several susceptible disulfide bonds in immunoglobulin (IgG) and human serum albumin (HSA) to form SCN adducts on one of the Cys components. We have now discovered a novel, endogenous, HSA Cys567-SCN adduct that is formed via CN reaction with the Cys558-- Cys567, C-terminal disulfide. Detection of a unique, 19-mer C-terminal peptide released by base hydrolysis of those HSA molecules containing the adduct is readily accomplished by liquid chromatography - mass spectrometry/mass spectrometry (LC-MS/MS). In plasma, reaction of CN with this HSA disulfide is rapid, and linearly related to the CN concentration at physiologically relevant levels. The adduct is very stable both to chemical treatment and to degradation by plasma enzymes. Quantitation of the adduct is achieved by integrated peak area comparison of a m/z 1272 daughter ion of the endogenous adduct with a deuterated (D6) peptide standard homologue (m/z 1278) using LC--MS-Selected Reactant Monitoring (LC-MS- SRM). The endogenous adduct is present in commercially available plasma and in the plasmas from 13 volunteers with an approximate 3-fold concentration difference between the highest and the lowest. Collectively, these results strongly suggest that the endogenous HSA Cys567-SCN adduct can serve as a retrospective biomarker of CN exposure. In this application, we propose to streamline our assay to reduce the time of sample preparation and to determine whether detection can be transferred from the LCQ platform, which we currently employ, to a quantitatively more precise and robust LC-triple quadrupole MS platform. Using the optimized assay, we will test the hypothesis that the quantity of the HSA Cys567-SCN adduct does not correlate with the free CN concentration that is present in whole blood. As part of our study, we will also compare control-matched samples from a cohort of volunteers with expected normal CN exposure to a cohort of firefighters. Firefighters are high risk for excessive CN exposure due to their proximity to industrial and residential fire smoke for prolonged periods. PUBLIC HEALTH RELEVANCE: The long-term health effects of acute, intermediate, and chronic cyanide exposure are poorly understood. Primary reasons for this are the rapid metabolism and clearance of cyanide and the inadequacy of current assays for cyanide, or its metabolites, to detect exposure accurately. Our proposed work is to test whether a novel, endogenous and readily detectable human serum albumin cyanide adduct (HSA Cys567-SCN) can serve as a retrospective biomarker of cyanide exposure. Using a MS-based assay developed by us to quantify the HSA Cys567-SCN adduct we will test whether adduct analysis can more accurately detect putative differences in cyanide exposure between control and high risk populations, such as firefighters, than currently used assays.